Isoprenoids (also known as terpenoids or terpenes) are a large, diverse group of complex natural products with considerable commercial interest. Isoprenoids are today mostly extracted from plants or chemically synthesized to be used as pharmaceuticals (e.g. taxol, bisabolol, lycopene, artemisinin), animal feed supplements and food colorants (various carotenoids) or flavours and fragrances (e.g. menthol, patchoulol, nootkatone). Isoprenoids are built from isoprene units (2-methyl-1,3-butadiene), and the biological precursor for all natural isoprenoids is isopentenyl diphosphate (IPP). Isoprenoids play important roles in living cells, such as in hormonal regulation (sterols), photosynthesis (carotenoids and others), just to mention a few.
In living cells, IPP can be generated by either the mevalonate-dependent (hereinafter MEV) pathway or the mevalonate-independent or MEP pathway, which are two distinct pathways. Most organisms rely exclusively on one of the pathways for generation of isoprenoid compounds, but plants and some microorganisms have both pathways. The precursor for synthesis of IPP via the MEV pathway is acetyl-CoA, which is synthesized in the central carbon metabolism. The precursors for synthesis of IPP via the MEP pathway are the glycolytic intermediates glyceraldehyde-3-phosphate (GAP) and pyruvate (PYR), which are synthesized in the central carbon metabolism. Both the mevalonate pathway and the MEP pathway produce dimethylallyl diphosphate (DMAPP) and IPP, and the reaction steps after IPP to produce geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are the same in cells using either the mevalonate pathway, the MEP pathway or both. It is from these intermediates that the various isoprenoid compounds in nature are produced.
The chemical industry, which produces organic molecules by traditional chemical processes, is increasingly turning to production processes utilizing microbial cell factories. The key drivers for this development towards green chemistry are that such so-called “biotechnological processes” are more environmentally friendly, that many compounds produced by microorganisms are too complex to be obtained by organic synthesis and that the microbial cell factory represents an unlimited supply of the particular compound. Currently, isoprenoids are produced at large scale by extraction from plants or by chemical synthesis. The major drawbacks of both of these methods are low yields and high costs. A third option is to produce the desired isoprenoids by in vitro enzymatic conversion, but this approach is limited by the availability of precursors, and therefore in most cases not economically viable.
Metabolic engineering of microorganisms for isoprenoid production may lead to production of large amounts of isoprenoids from cheap carbon sources in fermentation processes, and thereby solve many of the current problems in industrial isoprenoid production (Maury et al., 2005, Adv. Biochem. Engin/Biotechnol. 100:19), whilst allowing for biotechnological exploitation of the large diversity found in the isoprenoid group of natural compounds.
A number of isoprenoid products have been produced by genetically engineered microorganisms, including limonene (Carter et al., 2003, Phytochem. 64:425), carotenoids (Kajiwara et al, 1997, Biochem. J. 324: 421), epi-cedrol (Jackson et al, 2003, Org. lett 5: 1629), taxadiene (Huang et al, 2001, Biorg. Med. Chem 9: 2237), and others. In order to enhance isoprenoid production in E. coli, the genes dxs (encoding DXP synthase), dxr (DXP reductoisomerase) and idi (IPP isomerase) have been overexpressed with good results for several of the above-mentioned isoprenoid products (reviewed in Maury et al., 2005). A further increase in amorphadiene production by E. coli has been obtained by expressing the mevalonate pathway from S. cerevisiae in an amorphadiene producing strain of E. coli (Martin et al., 2003, Nat. Biotechnol. 21:796). Some isoprenoid compounds have also been produced in yeasts, including epi-cedrol in S. cerevisiae (Jackson et al., 2003, Org. Lett. 5:1629), lycopene and beta-carotene in S. cerevisiae (Yamano et al., 1994, Biosci. Biotechnol. Biochem. 58:1112) and Candida utilis (Miura et al., 1998, Appl. Environ. Microbiol. 64:1226). In several cases, isoprenoid production in yeast has been shown to be enhanced by overexpression of HMG1 (encoding HMG-COA reductase) (reviewed in Maury et al., 2005).
In spite of the background depicted above, there is still a need for further processes for producing terpenes and terpenoids and, in particular, for ways of accumulating terpenes in microorganisms with higher yields and in a less costly and time intensive manner than in the prior known methods. It is therefore an objective of the present invention to provide a method for producing terpenes or terpenoids that fulfils this need.
It is a further objective of the present invention to solve the problem of the supply of sufficient amounts of terpene precursors in an organism so as to enhance the accumulation of terpenes in the organism.
It is a particular objective of the present invention to produce a microorganism that accumulates and/or secretes high amounts of terpenes to the surrounding medium. The production of terpenes by such a microorganism is preferably stable over time.
Still a further objective underlying the present invention is to provide a biological platform for the production of terpenes, which is capable of producing any terpene at the choice of the manufacturer. Such a single system would in principle enable the production of one specific terpene at a time, but could be easily modified to produce another terpene or a mixture of terpenes. The same system could be used, of course, to produce different terpenes independently. Moreover, such a platform could also allow the production of terpene-derived compounds which are useful for the flavour and fragrance industry. One particular example which is not limitative of the invention is the production of nootkatone from valencene.
Furthermore, the biological production platform for terpenes of the invention preferably has high production capacity and is free of endotoxins and all the problems associated with them.
A further objective of the present invention is to provide a biological production platform highly capable of accumulating high levels of lipophilic compounds, even in a generally aqueous medium where such compounds, or their precursors, are not usually soluble.